npc natural product communications winning article.pdf · ken-ichi satoa, shoji akaia and juji...

Stereocontrolled Total Synthesis of Tetrodotoxin from myo-Inositol and D-Glucose by Three Routes: Aspects for Constructing Complex Multi-Functionalized Cyclitols with Branched-Chain Structures

Ken-ichi Satoa, Shoji Akaia and Juji Yoshimurab aDepartment of Material and Life Chemistry, Faculty of Engineering, Kanagawa University, 3-27-1, Rokkakubashi, Kanagawa-ku, Yokohama 221-8686, Japan

bDepartment of Basic Science, College of Science and Engineering, Iwaki Meisei University, Iwaki 970-8551, Japan

[email protected]

Received: March 30th, 2013; Accepted: June 8th, 2013

Reprinted with permission of the Natural Product Inc. Reference: Natural Product Communications, 8, 987-998 (2013)

This report describes the stereocontrolled total synthesis of the multi-functionalized cyclitol derivative, tetrodotoxin, containing eight asymmetric carbons and different types of branched-chains, from myo-inositol and D-glucose using three different methods. The tetrodotoxin derivatives possess a relatively small molecular weight but unique structural and chemical properties. Selection of the appropriate synthetic method may be useful not only for compounds related to TTX (including related derivatives), but also for other highly complex multi-functionalized cyclitols containing branched-chains.

Keywords: Tetrodotoxin, Functionalized cyclitol, Branched-chain sugar, Henry reaction, Ferrier(II) reaction. Tetrodotoxin (TTX 1), a well-known marine toxin, was originally isolated from the puffer fish [1]. Four groups reported the structure of TTX in 1964 [2-5]. The original name of TTX was “taricatoxin” (Figure 1).

Figure 1: Tetrodotoxin (1) and its analogs (1a-k).

TTX selectively interacts with elements of the sodium channel, inhibiting its activity in the cell membrane [6]. Therefore, TTX is employed as a tool to analyze events that occur via the sodium channel [7]. TTX has been isolated from the puffer fish, newts, frogs, octopi, crabs, shellfish, and numerous other aquatic animals. The animals do not produce TTX themselves; instead, it is produced by bacteria such as Alteromonas sp., Vibrio sp., and Shewanella sp. [8]. Yasumoto and others proposed a biosynthetic pathway of TTX and its analogs [9] based on the structure of TTX and its analogs [10]. TTX and its analogs are expected to provide important information in areas such as pharmaceuticals, structure-activity relationships, and biological roles [11]. Therefore, a facile and

large-scale synthesis of TTX and its analogs is of great interest. However, preparing modified TTX derivatives from the naturally occurring compound is difficult because of its unique structural and chemical properties (Figure 2) [12].

Figure 2: Chemical properties of tetrodotoxin.

The total synthesis of TTX and its analogs has presented difficult challenges, despite attempts by numerous research groups [13]. An efficient synthesis of optically active TTX has not been reported in the 30 years following the first total synthesis of (±) -TTX by Kishi et al. [14]. In 2003, the groups of Isobe and Du Bois succeeded in synthesizing ()-TTX, respectively [15a,15d]. Shortly afterward, Isobe’s group reported an improved route with fewer steps [15b,15c,15f-i]. In 2005, the total synthesis of (±) -TTX was achieved from myo-inositol [16a]; in 2008 and 2010 the synthesis of ()-TTX from D-glucose was accomplished [16b,16c]. Moreover, Noheda filed a patent application for synthetic methods for TTX and its analogs and intermediates [17]. A review on the synthetic work related to tetrodotoxin appeared in 2011 [18].

NPC Natural Product Communications 2015 Vol. 10 No. 5

691 - 702

692 Natural Product Communications Vol. 10 (5) 2015 Sato et al.

This report describes the methods and strategy for the stereocontrolled total synthesis of TTX using three different methods. For the synthesis of TTX, the use of carbohydrates as starting materials was considered for the following reasons. 1) They are inexpensive and readily available. 2) They have asymmetric carbons that can be used synthetically (for example, D-glucose can be used as a 5- or 6-membered compound to enable manipulation in a stereocontrolled manner to direct reaction at a specific position). The “chiron approach” [19] can also be employed in the total synthesis of complex natural products using chiral carbohydrates as a starting material. This approach has led to the stereocontrolled introduction of functionalized branched-chains into carbohydrate derivatives as chiral templates [20]. It is noteworthy that the method provides us with construction of a C-C bond bearing various functional groups at branching carbon atoms. Furthermore, the method of introduction allowed stereocontrolled construction of the functionalized branched-chain compounds without considering steric factors of the substrates. Based on these reports, universal methods for stereocontrolled construction of functionalized branched-chains onto carbohydrate derivatives have been developed (Figure 3 and 4) [21,22]. Stereocontrolled introduction of functionalized branched-chains into myo-inositol and D-glucose was performed using the developed methods. The Henry reaction (nitro-aldol reaction) [23] and the Ferrier(II) reaction [24] were employed for converting the branched-chain sugar into the corresponding branched-chain cyclitol. The Henry reaction has been investigated during synthetic studies of TTX [25] and the Ferrier(II) reaction has been examined for application to the synthesis of branched-chain cyclitol from branched-chain sugars [26]. Based on prior results, the total synthesis was achieved of naturally occurring branched-chain cyclitol compounds, such as cyclophellitol [27], mytilitol, and laminitol [28], starting from D-glucose. The synthetic strategies of TTX from carbohydrates using the three different methods A, B, and C are shown in Figure 5.

Method A: Stereocontrolled synthesis of (±) - tetrodotoxin from myo-inositol [16a] The total synthesis of (±) -TTX [29] from myo-inositol was achieved using an ortho ester as the protecting agent [30]. The synthetic strategy for (±) -TTX is shown as the retrosynthetic analysis in Figure 6. The (±)-TTX [29] can be considered chemically equivalent to compound A. The cyclic guanidine aminal may be constructed by neighboring group participation of the guanidine moiety and the aldehyde function. The ortho ester structure may be constructed by neighboring group participation of the carboxyl and axially oriented hydroxy groups. The aldehyde function of A can be introduced by oxidation of the hydroxymethyl group of B. The introduction of the di-N-Boc-guanidine moiety was planned for the last stage of the synthesis due to the instability of the intermediates, as anticipated from previous studies [14, 15]. The cyanohydrin group of B could be prepared by reaction of the -azido aldehyde function of C with a CN anion. Compound C could be synthesized from the corresponding carbonyl compound using dichloromethyl lithium by employing a previously reported method [21]. The asymmetric carbon atoms of TTX, C-5, C-7, and C-8, can be transformed into the C-5, C-3, and C-2 carbon atoms of myo-inositol, respectively. This important relation is maintained by the compulsory flip of the stable conformation into an unstable one while protected with an ortho ester. Following this outline, myo-inositol was modified using the following protection and deprotection steps (Scheme 1) [29]. Construction of the branched-chains at C-4 and C-6 of myo-inositol, and selective protection of the hydroxy groups occurred following a previously described procedure. Starting compound 3 was synthesized via orthoformate 2 from myo-inositol [30]. The equatorial hydroxy group of the mono-O-benzylated 3 was

Figure 3: Methods for constructing various functionalized branched-chain compounds.

OOHO

OO

O

CH3

OO

OO

O

HOO

O

CHCl2OO

O

LiO

OO

OO

O

OH

HO

OO

OO

O

O

OO

OO

O

OO

O

OO

O

HO OO

OO

O

OClO

O

OO

O

CHO

HO

OHCO

O

OO

O

OO

OO

O

HOO

O

OO

O

N3

OHC

c), i) i)

g)h)j)k)

g)c)l)

d) e) f)

a) b) c)

OOOHC

OO

O

OH

OO

OO

O

OH

OOHO

OO

O

HO

n)o)

Reagents and conditions: a) LDA, CH2Cl2, oxolane, -78 oC. b) H2O, rt. c) n-Bu3SnH, AIBN, toluene, reflux. d) 65 oC.

e) DBU, DMSO, rt. f) LiAlH4, oxolane. g) NaOH aq, 1.4-dioxane, reflux. h) NaOAc, 15-crown-5, HMPT, 70 oC.

i) NaBH4, aq MeOH, rt. j) NaBH4, DMSO, 80 oC. k) NaN3,15-crown-5, HMPT, 70 oC. l) NaOMe, HMPT, 70 oC.

m) n-Bu4NOH aq, DMSO, rt. n)CsOAc, 18-crown-6, toluene, reflux. o) NaBH4, MeOH, rt.

o)

l)

CHCl2

X

X

OMeOMe

ClClH

Preparation of tetrodotoxin from myo-inositol and D-glucose Natural Product Communications Vol. 10 (5) 2015 693

Figure 4: Complementary method for constructing functionalized branched-chains.

Figure 5: Synthetic strategies for TTX from carbohydrates using three different methods.

selectively protected by a methoxymethyl (MOM) group to give 4 in 74% yield. Compound 4 then was oxidized under Swern conditions to give the carbonyl compound 5 in quantitative yield. Addition of lithium diisopropylamide (LDA) and dichloromethane [21] gave the corresponding dichloroethanol derivative 6 as a single stereoisomer in 76% yield. Compound 6 was converted into unstable -hydroxy aldehyde derivative 7 [21], which was immediately reduced with NaBH4 without further purification to give the hydroxymethyl derivative 8 in good yield. The stereochemistry of the quaternary carbon atom in 6 and 8 was confirmed by NOE measurements, as shown in Figure 7. The complete benzylation of 8 to 9, followed by treatment with 0.1M

HCl-MeOH, gave the corresponding tetrol derivative 10 in 97% yield. The treatment of 10 with tert-butyldimethylsilyl chloride (TBDMS-Cl) and imidazole gave both the desired and the undesired mono-O-silylated compounds, 11 and 12, in 62% and 18% yields, respectively. The structures of 11 and 12 were confirmed by conversion into the corresponding acetates, 11a and 12a. Compound 12 was recycled to 11 by de- and reprotection via 10. The reaction of the cis-diol of 11 with 2,2-dimethoxypropane and a catalytic amount of pyridinium p-toluenesulfonate (PPTS) then gave the corresponding acetonide 13 in 93% yield. The conformation of 13 was confirmed by NMR analysis of the corresponding acetyl derivative 13a (J1,2 = 3.1 Hz, J2,3 = 7.3 Hz, J1,6 = 8.9 Hz, J6,5 = 3.1 Hz). Then, 13 was treated with dimethoxymethane and P2O5 to give the fully protected cyclitol derivative 14 in 75% yield. Removal of the benzyl group of 14 under catalytic reducing conditions gave 15 in 88% yield, which was then transformed into the acetonide 16 in 99% yield. The resulting alcohol 16 was oxidized to give the carbonyl compound 17 in 95% yield. Stereoselective introduction of the hydroxymethyl branched chain at the C-6 position of myo-inositol is important in this synthesis. Introduction of the hydroxymethyl branched-chain was first expected to be achieved from Wittig methylation [31] of the carbonyl compound 17 followed by hydroboration-oxidation. However, the alkene 19 was not obtained, likely due to steric hindrance of the MOM and/or

Figure 6: Retrosynthetic analysis of (± )-TTX (Method A). (a) The actual conformation of B is different: see compound 26. (b) One of the epimers is drawn.

Figure 7: NOE correlations of 6 and 8 [29].

TBDMS protecting groups. Peterson’s olefination [32] of 17 then was examined to give 19 via trimethylsilylmethyl compound 18 in 50% two-step yield. The methylene compound 19 was transformed into hydroxymethyl compound 20 in 74% two-step yield by


hydroboration-oxidation and de-silylation with tetra-n-butylammonium fluoride (TBAF). The conformation of 20 and its configuration at the hydroxymethyl branching carbon were confirmed by NMR analysis of its acetylated compound 20a (J5,6 = 5.8 Hz, J6,1 = 5.2 Hz) and 21 (J5,6 = 3.3 Hz, J6,1 = 3.3 Hz, and NOE experiments, shown in Scheme 1). The primary hydroxy

group of 20 was selectively protected with a tert-butyldiphenylsilyl (TBDPS) group to give the corresponding monohydroxy compound 22 in 87% yield. Compound 22 was then oxidized with Dess-Martin periodinane [33] to give 23 (a key precursor for synthesizing TTX) in 99% yield (Scheme 1).

R1O

OR3

OBn

OR4

OR2

BnO

BnO

O OOR1

OO

R3OOR2

O OOR1

HOHO

HO

OR2

O OOR1

BnOBnO

BnO

OR2

BnOR1O

OO

OOR

R1O

10: R = H10a: R = Ac

11: R1 = R3 = R4 = H, R2 = TBDMS11a: R1 = R3 = R4 = Ac, R2 = TBDMS12: R1 = R2 = R4 = H, R3 = TBDMS12a: R1 = R2 = Ac, R3 = TBDMS, R4 = H

9: R = MOM, R1 = Bn8: R = MOM, R1 = H

13: R1 = H, R2 = TBDMS

14: R1 = MOM, R2 = TBDMS

13a: R1 = Ac, R2 = TBDMS

15: R1 = MOM, R2 = TBDMS

16: R1 = MOM, R2 = TBDMS, R3 = H

10

16a: R1 = MOM, R2 = TBDMS, R3 = Ac

RO

OR

OBn

OR

OR

BnO

BnO

(COCl)2, DMSO,Et3N / CH2Cl2 (95%)

15

3

NaH, BnBr/ DMF (89%)

0.1M HCl-MeOH(97%)

Ac2O, Py. (quant.)

TBDMS-Cl, imidazole / DMF(11: 62%; 12: 18%)

70% AcOH (quant.)Ac2O, Py. (quant.)

Ac2O, Py. (quant.)

Me2C(OMe)2, PPTS /CH2Cl2 (93%)

10% Pd(OH)2-C,H2 / EtOH (88%)

CH2(OMe)2, P2O5

/ CH2Cl2 (75%) Ac2O, Py. (quant.)

Me2C(OMe)2, PPTS/ CH2Cl2 (99%)

Ac2O, Py. (quant.)

OO

OR3

OO

OR1

OR2

OO

OH

OO

OR

OTBDPS

O O

OO

CH2TMS

OTBDMS

O OOR

OO

CH2

OTBDMS

17: R = MOM 19: R = MOM

18: R = MOM

20: R1 = MOM, R2 = R3 = H20a: R1 = MOM, R2 = R3 = Ac

23: R = MOM22: R = MOM

OROR

RORO

OR

OR

OR

HH

NOE

21: R = Ac

7.3%

5

13

1) BH3-THF, aq NaOH,then aq H2O2

2) n-Bu4NF / CH2Cl2(2 steps 74%)

5

13

[Ph3P=CH3]+Br ,

n-BuLi / THF

TMSCH2MgCl/ THF (63%)

NaH / THF(79%)

NaBH4 /EtOH (95%)

1) 70% aq AcOH2) Ac2O, p-TsOH

(2 steps quant.)

Ac2O, Py. (quant.)

TBDPS-Cl,imidazole /CH2Cl2 (87%)

Dess-Martinperiodinane /CH2Cl2 (99%)

OR OH

OO O

OO

OR

OTBDPS

O OOR

OO

O

OTBDMS5

3

1

BnO

OO

O

O

ORHO

HO

HO

OH

OHHO

R1OHO

OO

OOR2

myo-Inositol

meso-2: R1 = R2 = H

3: R1 = Bn, R2 = H

4: R1 = Bn, R2 = MOM

5: R = MOM

5

3 1CH(OEt)3, p-TsOH/ DMF (70%)

NaH, BnBr / DMF (81%)

MOM-Cl, i-Pr2NEt / CH2Cl2 (74%)

(COCl)2, DMSO,Et3N (quant.)

BnOHO

OO

OOR2

R1

6: R1 = CHCl2, R2 = MOM

7: R1 = CHO, R2 = MOMn-Bu4NOH / Me2SO

LDA, CH2Cl2 (76%)NaBH4 / MeOH(2 steps 81%)

Scheme 1: Synthesis of key intermediate 23 from myo-inositol via branched-chain cyclitol derivative 8 on the path to ( ± ) - TTX (with myo-inositol numbering) [29].

Scheme 2: Synthesis of precursor 32 from 23 on the path to ( ± ) - TTX (with muco-inositol numbering) [29].

26a: R = H 26: R = H27: R = MOM

OO

OO

OR

OTBDPS

O OO

OO

OR

TBDPSO

O

Cl

N3

OO

OO

OR

OTBDPS

OH

CHCl2H

H

23: R = MOM24: R = MOM

15

NOE 5.5%NOE 4.7% 25: R = MOM24a: R = MOM

6'

MOMO

O

N3

OOTBDPS

O

O CN

OR

MOMO

O

N3

OOTBDPS

O

O CN

OR

13

5

OO

OO

OR

OTBDPS

CHO

13 5

LDA, CH2Cl2/ THF (79%)

NaN3, 15-crown-5/ DMSO (63%)

TMS-CN, Et3N / MeOH(26: 45%; 26a: 29%)

TMS-CN, Et3N / MeOH(26: 44%; 26a: 25%)

CH2(OCH3)2, P2O5/ CH2Cl2 (93%)

O

N3

OO

MOMO

OTBDPS

CHO

OMOM

O

OO

OO

O

OMOM

N3

OTBDPS

OH

OO

OO

O

OMOM

NH2

OH

O

NH

NBoc

NHBoc

OO

OO

O

OMOM

OR1

O

OO

OO

O

OMOM

X

OTBDPS

O

29: X = N3

30: X = NH2

28

32: R1 = H32a: R1 = Ac

531

531

31

DIBAL-H/ CH2Cl2 (87%)

CrO3 in aq H2SO4

/ CH2Cl2-acetone (90%)

10% Pd-C, H2 / EtOH (quant.)

TBAF / THF (90%)

(BocNH)2C=S, HgCl2,Et3N / DMF (72%)

Ac2O, Py. (quant.)

13

5

4a68a

N3

with TTX numbering

3

66

6' 6'

6 6


Introduction of amino function and ortho ester moieties of B and A was accomplished as follows (Scheme 2) [29]. To construct the -azido aldehyde branched chain of C, 23 was treated with LDA and dichloromethane at 78C. The expected dichloroethanol derivative 24 was obtained in 79% yield [21]. The stereochemistry at C-6 of 24 was determined by NOE experiments, together with results from the stereoselective reduction of 23 to 22 (Scheme 1, 2). Hence, ketone 23 was reduced with NaBH4 to give only corresponding axial alcohol 22. The stereoselectivity of the nucleophilic reaction of 23 may be controlled by the 1,3-diaxial relationship of the C-3 and C-5 substituents. The resulting dichloroethanol derivative 24 was then treated with NaN3 in 15-crown-5 ether and DMSO to give the corresponding -azido aldehyde 25 in 63% yield [21]. The reaction proceeded both stereoselectively and regioselectively. The stereochemistry at C-6 of 25 was verified by X-ray crystallography of cyanohydrin derivative 26 (Figure 8), which was synthesized by reaction of 25 with CN anion as follows. Reaction of -azido aldehyde 25 and TMS-CN/Et3N in MeOH gave the corresponding epimeric cyanohydrin derivative 26 [34] and 26a in 45% and 29% yields, respectively. Careful monitoring of the cyanohydrine reaction by TLC (n-hexane-EtOAc, 6:1) suggested that 25 was completely converted into the kinetically controlled product 26a within 3 min, after which it gradually converted into an equilibrium mixture (3:2) of 26 and 26a. The more polar product 26 was then treated with dimethoxymethane and P2O5 to give the corresponding protected methoxymethyl (MOM) derivative 27 in 93% yield. In contrast, the less polar isomer 26a could not be protected under these conditions. The undesired isomer 26a could be transformed into desired isomer 26 by treatment with TMS-CN/Et3N in MeOH in 44% yield (Scheme 2). The configurations at C-6 and C-6’ (C-8a and C-9 with TTX numbering) of 26 were determined by single-crystal X-ray crystallography [34a]. The ORTEP diagram of 26 shows that the configuration of the six-membered ring is the 3C6 form and the configuration at C-6’ (C-9 with TTX numbering) is the same as that of natural TTX (Figure 8). Figure 8: ORTEP diagram of 26 with the muco-inositol based atomic numbering [19a]. Thermal ellipsoids are drawn at the 30% probability level. Introduction of ortho ester moiety and guanidine groups to 27 was accomplished as follows (Scheme 2) [29]. Cyanohydrin derivative 27 was treated with diisobutylaluminum hydride (DIBAL-H) to give the corresponding aldehyde derivative 28 in 87% yield. The selective deprotection of the MOM group at O-4 (O-5 with TTX numbering) of 28 and subsequent treatment with Jones’ reagent gave the -lactone derivative 29 as a single product in 90% yield. The structure of 29 was confirmed by NMR analysis [W-shaped long-range coupling: J5,6’ (J4a,9 with TTX numbering) = 1.4 Hz,

NOESY, and HMBC experiments], as shown in Figure 9. To introduce the guanidine moiety, the azido group of 29 was first reduced with 10% Pd-C and H2 to quantitatively give amino derivative 30. Then, the TBDPS group of 30 was deprotected using TBAF in 90% yield. Thus, amino derivative 31 was then treated with bis(tert-butoxycarbonyl)thiourea, mercury(II) chloride, and triethylamine [35] to give the guanidine derivative 32 in 72% yield. The structure of 32 was confirmed by conversion to the corresponding acetate 32a.

29: R = MOM

1

3

HMBC

O

OO

O

H

N3

OTBDPSH

O

OOR

H

H

W-coupling

NOESY

5

J5,6' 1.4 Hz(J4a,9 with TTX numbering)

(between carbonyl carbon and H-5)

6'

Figure 9: NOE correlations of 29 (with muco-inositol numbering) [29].

Finally, oxidation of 32 with pyridinium chlorochromate (PCC) and subsequent treatment of the product with 4M HCl-dioxane/MeOH [15a,b], then 30% TFA solution, followed by a 4% aq. AcOH solution, provided a mixture of (±) -TTX, 4,9-anhydro-4-epi-(±) - TTX ((±) -anhydroTTX), and other polar decomposition compounds. The mixture was purified by HPLC on a Hitachi-gel #3013-c column (H+ form, 0.05M aq. AcOH) [15a] to give (±) -TTX (including a sight lactone form by 1H NMR), and (±) -anhydroTTX in 30% and 10% yields (from 32), respectively (Scheme 3). Isomerization of the (±)-anhydroTTX to (±) -TTX in 4% aq. AcOH solution was very slow at temperatures below room temperature. The spectral data for the synthetic (±)-TTX were identical with those of natural TTX [36] (1H and MS) [9d], [15a,b] (The comparative 1H NMR data are illustrated in Table 1 of Method B).

Scheme 3: Final conversion of precursor 32 into ( ± ) - TTX [29]. In the oxidation reaction described above, the signals in the 1H NMR spectrum of the oxidation product were broadened, which made the confirmation of the original aldehyde signals impossible. The results suggested that resulting aldehyde 33a was interconvertible with the aminal compound 33b. A similar phenomenon has been reported for 1,2-O-isopropylidene-3-C-aceta- minoethyl--D-xylo-pentodialdofranose [20a]. Treatment of the other oxidation products (Dess-Martin [33], Swern, or 2,2,6,6-tetramethyl-1-piperidinyloxy radical (TEMPO) [37] oxidation products) under acidic conditions (in Scheme 3) did not provide either (±) -TTX or (±)-anhydroTTX. Some side reactions must


have occurred during oxidation of the primary hydroxy group of 32. These oxidation products showed different Rf values on TLC compared with those of 33a (33b). The determination of its structure was difficult because of this broadened 1H NMR spectrum. In conclusion, the total synthesis of ( ± ) -TTX from myo-inositol was accomplished in a highly stereocontrolled fashion and in excellent yield (34 steps to 1 from myo-inositol). Key steps included 1,3,5-orthoformylation of myo-inositol with conformational inversion to differentiate the remaining three free OH groups. These groups were oxidized in a stepwise manner to the carbonyl function, followed by specific nucleophilic addition, including successive spiro -chloroepoxide formation and its ring-opening with an azide anion, to give the desired branched-chain structure. Method B: Stereoselective and efficient total synthesis of optically active tetrodotoxin from D-glucose [16b]: The total synthesis of optically active ()-TTX from D-glucose was achieved using the Henry reaction [23] as the key transformation. The synthetic strategy for ()-TTX is shown as a retrosynthetic analysis in Figure 10. The retrosynthetic plan for the synthesis of ()-TTX from D-glucose focuses on preparation of the key intermediate, ketone D (23). This intermediate was successfully converted to TTX [16a] as mentioned in method A (Scheme 1-3). In method A, (±) -TTX was synthesized via compounds A’, B’, C’, and D. Therefore, the synthesis of optically active compound D from D-glucose was a promising route to the synthesis of ()-TTX. Compound D can be synthesized from compound E through the Henry reaction, as previously described [25]. The key intermediate, E, was synthesized using standard reactions from D-glucose, as described below (Scheme 4-6). 3-C-Hydroxymethyl-1,2:5,6-di-O-isopropylidene--D-gluco-hexofuranose (36) [20a] was prepared by stereoselective m-chloroperbenzoic acid (m-CPBA) oxidation of 3-deoxy-1,2:5,6-di-O-isopropylidene-3-C-methylene--D-ribo-hexofuranose (34) [38] derived from D-glucose, followed by hydrolysis with 1M aq. NaOH. The two hydroxy groups of 36 were protected with a benzyl (Bn) group to give 3,3’-di-O-benzyl derivative 37 in 95% yield. Compound 37 then was selectively hydrolyzed using 70% aq. AcOH solution at room temperature to give the 5,6-diol 38 in quantitative yield. Oxidative degradation of 38 with NaIO4 in aq. MeOH solution gave the corresponding aldehyde 39, which then was treated with nitromethane in methanol in the presence of NaOMe to give nitro alcohol 40 (diastereomer ratio = ca. 10:1) in 75% two-step yield. The diastereomeric mixture of nitro alcohol 40 was treated with methanesulfonyl chloride in the presence of triethylamine to give the corresponding single nitro-olefin 41 (G in Figure 10) in 85% yield. Reaction of 41 with n-butyl lithium and bis(phenylthio)methane gave the branched bis(phenyl)dithioacetal derivatives 42a and 42b. The ratio of 42a:42b (ca. 10:1) was determined by 1H NMR (comparison of the intensities of H-5) of the crude mixture. The addition of dithioacetal anion proceeded with greater stereoselectivity (10:1) than that described in a previous report [25a] using 1,3-dithiane (5:4). The greater stereoselectivity might be due to the bulkiness of the bis(phenylthio)methane reagent. The desired compound 42a was isolated by fractional crystallization in 80% yield. The configuration at C-5 of 42a was determined by derivation into the corresponding cyclitol compound 44a, as described below. The 1,2-O-isopropylidene group of 42a was hydrolyzed upon treatment with 85% aq. AcOH solution under reflux. The resulting nitro-sugar 43 (F in Figure 10) was treated with 1.5 mol eq of NaHCO3 in aq. MeOH solution to give crystalline muco-inositol derivative 44 [39] (E in Figure 10) in 84% two-step yield.

Figure 10: Retrosynthetic analysis of ()-TTX from D-glucose (Method B). (a) The actual conformation of B’ is different: see compound 26.

Acetylation of 44 under standard conditions gave tri-O-acetate 44a, quantitatively. The coupling constant for 44a (J1,2 = 2.5 Hz, J4,5 = 3.5 Hz, J1,6 = J5,6 = 11.0 Hz) suggested the configuration for C-5 (C-8 with TTX numbering). The formation of four stereoisomers, including 44, is possible from the Henry reaction, but the three undesirable stereoisomers were not isolated. This stereoselectivity is likely to be due to the stable six-membered cyclic transition state of intermediate [40]. Reaction of the vicinal-diol of 44 with 2,2-dimethoxypropane and a catalytic amount of p-toluenesulfonic acid (p-TsOH) gave the corresponding acetonide 45 in 86% yield. Acetonide 45 then was treated with dimethoxymethane and P2O5 to give the fully protected cyclitol 46 in 93% yield. Treatment of 46 with N-bromosuccinimide (NBS) in aq. MeCN solution gave the unstable aldehyde 47, which was immediately reduced with NaBH4 to give the hydroxymethyl derivative 48 in 82% two-step yield. Silylation of 48 with TBDPS-Cl and imidazole gave the fully protected nitro cyclitol 49 in 95% yield (Scheme 5). Transformations of the nitro derivative into the carbonyl derivative were examined. Initially, compound 49 was treated with potassium tert-butoxide (t-BuOK) and m-CPBA in benzene [41a] to give decomposed polar compound(s) and a minor amount of the desired product 50 (ca. 5% yield). Some side reactions (mainly retro-aldol reaction and subsequent decomposition under basic conditions) proceeded during oxidation of the nitronate intermediate. Therefore, McMurry’s transformation [41b] from the nitronate to the carbonyl was applied. Compound 49 was treated with t-BuOK, and the resulting nitronate was subjected to ozonolysis (O3) in toluene at


Scheme 4: Synthesis of nitro cyclitol 44 (E) using the inter- and intra-molecular Henry reaction.

–78C to give ketone 50 in moderate yield (79%). The transformation of 50 into compound 23 (D in Figure 10) was achieved by catalytic reduction, and subsequent acetonide protection of the resulting diol in 79% two-step yield (Scheme 5). Further conversion of ketone 23 into ()-TTX was conducted using an established route for ( ± ) -TTX synthesis, including successive spiro -chloroepoxide formation and its ring-opening with azide anion [21, 22] as a key step. To obtain compound 25, ketone 23 was treated with LDA and dichloromethane at –78C to give the expected dichloroethanol derivative 24 as a single isomer in 79% yield. Dichloroethanol derivative 24 was treated with NaN3 and 15-crown-5 ether in DMSO at 75C to give the -azido-aldehyde 25 (C’ in Figure 10) via spiro -chloroepoxide derivative 24a in 64% yield, with complete stereo- and regio-selectivity. The structure of 25, including the stereochemistry at C-6 (C-8a with the corresponding TTX numbering), was determined by X-ray structure analysis. The reaction of -azido-aldehyde 25 and TMS-CN/Et3N in MeOH gave the corresponding cyanohydrin 26 and its epimer 26a in 56% and 17% yields, respectively. Stereochemistry at C-6’ (C-9 with the corresponding TTX numbering) of 26 was the same as that observed in the total synthesis of (±) -TTX. The undesired isomer 26a could be isomerized into the desired isomer 26 under the same reaction conditions. After two cycles of this procedure, the total yield of 26

Scheme 5: Synthesis of optically active key intermediate 23 (D).

Scheme 6: Conversion of optically active ketone 23 (D) into ()-TTX (1) (with muco-inositol numbering).


was 68%. To introduce amino and ortho ester moieties, the hydroxyl group of 26 was protected with the methoxymethyl (MOM) group, and subsequently treated with DIBAL-H to give the corresponding aldehyde 28 in 82% two-step yield from 26. Selective deprotection of the MOM group at O-4 (O-5 with the corresponding TTX numbering) of 28 and subsequent treatment with Jones’ reagent gave the -lactone 29 as a single product in 90% yield. Monitoring of the reactions of aldehyde 27 using TLC (n-hexane/ethyl acetate 4:1) suggested that the deprotection of the MOM group first formed the hemiacetal intermediate, then immediately oxidized into -lactone 29. To introduce the guanidine moiety, the azido group of 29 first was reduced with 10% Pd-C and H2 to give the amine 30 in quantitative yield. Subsequent deprotection of the TBDPS group using TBAF gave alcohol 31. Guanidinylation [35] of 31 provided the precursor 32 (A’ in Figure 10), which was converted to 4,9-anhydro-4-epi-TTX (anhydroTTX, 1a) by oxidation with pyridinium chlorochromate (PCC), followed by treatment with 4M HCl-dioxane/MeOH, [15a,b] and then 30% aq. CF3COOH in 50% yield. Isomerization of 1a by treatment with 4% aq. AcOH at 60C for two days yielded a 1:4 mixture of 1a:1. The mixture was purified by HPLC [15a] to give 1 in 56% yield (Scheme 6). The spectral data (1H, MS) for the synthetic 1 agreed well with those of natural TTX [9d,15a,b,36] (Table 1 and Figure 11). Moreover, specific rotations of synthetic 1a and 1 were []28

D +1.2 (c 0.18, 3%

aq. AcOH) and []28

D –3.75 (c 0.13, 3% aq. AcOH), respectively. Both rotation values agreed well with those reported by Isobe et al. [15a-c,e]. Table 1: Comparative 1H NMR spectroscopic data of ()-TTX (1) [9f].

(a) 600 MHz, in 3% CD3COOD/D2O, referenced to CHD2COOD (2.06 ppm).

Figure 11: 1H NMR spectra of ()-TTX (1) and ()-anhydroTTX. In conclusion, the total synthesis of optically active ()-TTX was accomplished from D-glucose using the Henry reaction as a key framework in a highly stereoselective manner (34 steps to 1 from D-glucose). Method C: Total synthesis of ()-tetrodotoxin from D-glucose: A route to multi-functionalized cyclitol using the Ferrier(II) reaction [16c]: Another route was used for synthesizing ()-TTX from D-glucose using the Ferrier(II) reaction [24] for key skeleton construction. The synthetic route to ()-TTX was based on prior experience with the total synthesis of (±)-TTX from myo-inositol [16a]. Total syntheses

using methods A and B were conducted successfully using the common intermediate D (23), eventually in an enantiospecific manner [16b]. The synthetic strategy for ()-TTX is shown as a retrosynthetic analysis in Figure 11. Compound D (23) was derived from D-glucose through multi-functionalized cyclitol I [16a,b], which can be prepared by reaction of enol acetate J via the Ferrier(II) reaction [24]. Compound J was obtained by standard transformations of D-glucose using stereoselective introduction of a branched chain at C-3 with D-glucose (C-6 with the corresponding TTX numbering). The transformation of D into ()-TTX, via compounds C’, B’, and A’, was accomplished in a manner similar to that used for methods A and B [16a,b].

Figure 12: Retrosynthetic analysis of ()-TTX from D-glucose (Method C). (a) The actual conformation of B’, I, and H is different: see compound 26, 59a, and 61 with the corresponding TTX numbering. The total synthesis of ()-TTX from D-glucose is shown in Schemes 7 and 8. Methyl 2-O-benzyl-4,6-O-benzylidene--D-glucopyranosid-3-ulose (51) was prepared by regioselective benzylation followed by Swern oxidation of methyl 4,6-O-benzylidene--D-glucopyranoside derived from D-glucose. Peterson’s olefination [42] of 51, followed by treatment with aq. AcOH solution, gave methylene derivative 52 via a trimethylsilyl derivative. Regioselective protection of 52 with pivaloyl and methoxymethyl groups gave 54, which was oxidized using m-CPBA via a stereochemical course reported previously [43] to afford epoxide 55. Alkaline hydrolysis of 55 yielded triol 56, which was then converted into the acetonide 57. Oxidation of 57 resulted in an unstable aldehyde, which was immediately converted into enol acetate 58 (J in Figure 12) as a single geometrical isomer. The (Z)-configuration at C-8a of 58 was determined by NOESY analysis


(Figure 13). The key reaction of the intramolecular cyclization [Ferrier(II) reaction] of enol acetate 58 involved several mercury reagents in aq. AcOH-acetone solution at room temperature. Results are shown in Table 2. As a result, the desired stereoisomer 59a (I in Figure 12) was obtained in 58% yield using Hg(OAc)2 (59a:59b:59c:59d = 6:2:1:0) under the conditions listed in entry 4. In contrast, Ferrier(II) reaction using PdCl2 [24b] afforded a complex mixture. The (-allyl)palladium complex reaction intermediate was not smoothly generated, and the starting enol acetate gradually decomposed during heating. Thus, Hg(OAc)2 was chosen as the most suitable reagent for this enol acetate 58. Structures of the four stereoisomers 59b-d were confirmed by 1H NMR analysis. The chemical shifts and J values of 59a suggested the structure in Figure 13 (H-8a, 5.21, J8a,8 = 3.3 Hz; H-8, 4.21, J8,7 = 2.2 Hz, J8,OH = 1.5 Hz; H-4, 4.02, J7,5 = 0 Hz; H-5, 4.31, J8a,5 = 1.0 Hz). Furthermore, the configurations at C-6 and C-8a of 59a with the corresponding TTX numbering were verified by single-crystal X-ray crystallography of 61 (Figure 14). Efforts to transform 59b-d

Scheme 7: Synthesis of multi-functionalized cyclitol derivative 59 toward ()-TTX using the Ferrier(II) reaction (with the corresponding TTX numbering). Table 2: Ferrier(II) reaction conditions and product yields.

Figure 13: Structural assignments of enol acetate 58 and cyclitol 59a (chemical shifts, spin-spin coupling pattern, NOE correlation of 58 and J values of 59a with the corresponding TTX numbering).

Scheme 8: Total synthesis of ()-TTX from multi-functionalized cyclitol derivative 59a (with the corresponding TTX numbering).

Figure 14: ORTEP drawing of 61 with the corresponding TTX numbering. Thermal ellipsoids were scaled to enclose 30% probability.

into 59a were unsuccessful. Some side reactions, such as -elimination, occurred during the stereochemical inversion of hydroxy substituents at C-8 (Scheme 7). Protected diol 60 was obtained by catalytic debenzylation of 59a, followed by isopropylidenation. Compound 60 was then transformed into 61 (H in Figure 12) by Peterson’s olefination [42] and subsequent O-silylation. Methylene derivative 61 was converted into the corresponding hydroxymethyl derivative 62 by hydroboration-oxidation and de-O-silylation. Selective protection of the primary hydroxy group of 62 using TBDPS group afforded the corresponding monohydroxy compound 63, which was then oxidized with Dess-Martin periodinane [33] to give optically active ketone 23 (D in Figure 12), a key intermediate in the synthesis of TTX (Scheme 8). Further reactions of 23 transformed it into ()-TTX along an established route [16a], including successive spiro -chloroepoxide formation and its ring-opening with an azide anion [21,22] as a key step. Spectral data (1H and MS) for the synthetic TTX agreed well with those for natural TTX [15a,b,16a,b] (same as method B). The total synthesis of ()-TTX from D-glucose using the Ferrier(II) reaction as the key skeleton construction reaction was achieved successfully using effective and stereoselective steps (35 steps to 1 from D-glucose).

In studies of TTX synthesis, the total synthesis of TTX by three routes (method A, B, and C) was accomplished [16a,16b,16c]. These methods provide a choice for synthesizing compounds related


to TTX (including related derivatives), as well as other highly complex multi-functionalized cyclitols bearing branched-chain structures such as cyclophellitol [27], mytillitol, and laminitol [28]. Acknowledgments - The authors thank Dr T. Nakagawa, Professor Emeritus of Yokohama City University, Dr H. Hashimoto, Professor Emeritus of Tokyo Institute of Technology, and Dr M. Funabashi, Professor Emeritus of Ciba University, for helpful

discussions. The authors also thank Dr Y. Nakamura, Dr Y. Kajihara, Dr K. Suzuki, and Mr N. Sugita. This work was partially supported by “High-Tech Research Center Project” and "Science Frontier Project of Kanagawa University" and Grants-in-Aid for Scientific Research (No. 01540460, 19750147) from the Ministry of Education, Science, Sports and Culture, Japan.

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“Chiron” Approach. Organic Chemistry Series. Vol. 3, Baldwin JE. (Ed.), Pergamon Press, Oxford, UK, 21-26. [20] (a) Yoshimura J, Kobayashi K, Sato K, Funabashi M. (1972) Configuration of branched-chain derivatives of 1,2:5,6-di-O-isopropylidene-α-D-ribo-

hexofuranos-3-ulose. Bulletin of the Chemical Society of Japan, 45, 1806-1812; (b) Yoshimura J, Mikami K, Sato K, Shin C. (1976) Branched-chain sugars. VIII. On the configuration of branched-chain sugars from methyl 3-O-benzoyl-4,6-O-benzylidene-α-D-arabino-hexopyranosid-2-ulose. Bulletin of the Chemical Society of Japan, 49, 1686-1689; (c) Yoshimura J, Sato K. (1978) Branched-chain sugars. XII. The stereoselectivities in the reaction of methyl 4,6-O-benzylidene-α- and -β-D-hexopyranosid-3-uloses with diazomethane. Bulletin of the Chemical Society of Japan, 51, 2116-2121; (d) Yoshimura J, Sato K, Funabashi M. (1979) Branched-chain sugars. XVII. Stereoselectivity in the oxidation of several methyl 4,6-O-benzylidene-2-C- or -3-C-methylene-α- and -β-D-hexopyranosides with m-chloroperbenzoic acid. Bulletin of the Chemical Society of Japan, 52, 2630-2634; (e) Yoshimura J, Sato K. (1979) Branched-chain sugars. Part XIII. Stereoselectivities in the reaction of methyl 4,6-O-benzylidene-α- and -β-D-hexopyranosid-2-uloses with diazomethane. Carbohydrate Research, 73, 75-84; (f) Matsuzawa M, Sato K, Yasumori T, Yoshimura J. (1981) Branched-chain sugars. XXIII. Stereoselectivities in the addition of nucleophiles to several 4-uloses. Bulletin of the Chemical Society of Japan, 54, 3505-3509; (g) Yoshimura J, Sato K. (1983) Branched-chain sugars, part XXXIII. Complementary stereoselectivity in the reactions of hexopyranosid-4-uloses with methylmagnesium iodide and methyl lithium. Carbohydrate Research, 123, 341-346.

[21] (a) Sato K, Suzuki K, Ueda M, Katayama M, Kajihara Y. (1991) A novel reagent for the synthesis of branched-chain functionalized sugars. Dichloromethyl lithium. Chemistry Letters, 1469-1472; (b) Sato K, Suzuki K, Ueda M, Kajihara Y, Hori H. (1997) Useful intermediates, 3-C-dichloromethyl furanose derivatives, for the synthesis of branched-chain functionalized sugars. Bulletin of the Chemical Society of Japan, 70, 225-230.

[22] Sato K, Sekiguchi T, Hozumi T, Yamazaki T, Akai S. (2002) Improved synthetic method for preparing spiro α-chloroepoxides. Tetrahedron Letters, 43, 3087-3090.

[23] For reviews of the Henry reaction, see: Luzzio FA. (2001) The Henry reaction: recent examples. Tetrahedron, 57, 915-945. [24] For reviews of the Ferrier (II) reaction, see: (a) Ferrier RJ, Middleton S. (1993) The conversion of carbohydrate derivatives into functionalized

cyclohexanes and cyclopentanes. Chemical Reviews, 93, 2779-2831; (b) Takahashi H. (2002) Synthetic studies on sugar-related bioactive substances based on the ring transformation. Yakugaku Zasshi, 122, 755-771; (c) Bender SL, Budhu RJ. (1991) Biomimetic synthesis of enantiomerically pure D-myo-inositol derivatives. Journal of the American Chemical Society, 113, 9883-9885.

[25] (a) Funabashi M, Wakai H, Sato K, Yoshimura J. (1980) Branched-chain sugars. XV. Synthesis of 1L-(1,2,3’,4,5/3,6)-3-hydroxumethyl-4,5-O-isopropylidene-3,3’-O-methylene-6-nitro-2,3,4,5-tetrahydroxy-cyclohexanaldehyde dimethylacetal-potential key compound for total synthesis of optically active tetrodotoxin. Journal of Chemical Society, Perkin I, 14-19; (b) Sato K, Kajihara Y, Nakamura Y, Yoshimura J. (1991) Synthesis of functionalized cyclohexanecarbaldehyde derivative. A potential key compound for total synthesis of optically active tetrodotoxin. Chemistry Letters 1559-1562

[26] Sato K, Sakuma S, Nakamura Y, Kajihara Y, Yoshimura J. (1991) Synthesis of methyl-branched 2,3,4,5-tetrahydroxycyclohexanone derivatives via 6-deoxyhex-5-enopyranosides. Chemistry Letters, 17-20.

[27] Sato K, Bokura M, Moriyama H, Igarashi T. (1994) Total synthesis of a novel β-glucosidase inhibitor, cyclophellitol starting from D-glucose. Chemistry Letters, 37-40.

[28] Sato K, Bokura M, Taniguchi M. (1994) Syntheses of optically active 2,3,6-tri-O-benzyl-D-myo-inositol, laminitol, and mytilitol from D-glucose. Bulletin of the Chemical Society of Japan, 67, 1633-1640.

[29] All intermediates were racemic unless otherwise noted. [30] (a) Lee HW, Kishi Y. (1985) Synthesis of mono- and unsymmetrical bis-orthoesters of scyllo-inositol. Journal of Organic Chemistry, 50, 4402-

4404; (b) Billington DC, Baker R. (1987) Synthesis of myo-inositol 1,3,4,5-tetraphosphate and myo-inositol 1,3-bisphosphate. Journal of the Chemical Society, Chemical Communications, 1011-1013; (c) Baudin G, Glazer BI, Swaminathan KS, Vasella A. (1988) A synthesis of 1D- and 1L-myo-inositol 1,3,4,5-tetraphosphate. Helvetica Chimica Acta, 71, 1367-1378.

[31] Inch TD, Lewis GJ. (1972) Synthesis and degradation of benzyl 4,6-O-benzylidene-2,3-dideoxy-3-C-ethyl-2-C-hydroxymethyl--D-glucopyr anoside and -mannopyranoside. Carbohydrate Research, 22, 91-101.

[32] Chan TH, Chang E, Vinokur EM. (1970) The synthesis of olefins and ketones from carbonyl compounds and carbanions alpha to silicon. Tetrahedron Letters, 14, 1137-1140.


[33] (a) Dess DB, Martin JC. (1991) A useful 12-I-5 triacetoxyperiodinane (the Dess-Martin periodinane) for the selective oxidation of primary or secondary alcohols and a variety of related 12-I-5 species. Journal of the American Chemical Society, 113, 7277-7287; (b) Dess DB, Martin JC, (1983) Readily accessible 12-I-5 oxidant for the conversion of primary and secondary alcohols to aldehydes and ketones. Journal of Organic Chemistry, 48, 4155-4156.

[34] (a) Akai S, Nakamura Y, Kajihara Y, Yoshimura J, Sato, K. (1999) Crystal structure of potential key compounds, DL-(1,2,3',4,5,6'/3,6)-6-azido-5'-O-t-butyldiphenylsilyl-6-(cyano)hydroxymethyl-3,5-di-C-hydroxymethyl-1,2:3,3'-di-O-isopropylidene-4-O-methoxymethyl-1,2,3,4-cyclohexanetetrol, toward (±)-tetrodotoxin. Analytical Sciences, 15, 503-504; (b) Sato K, Kajihara Y, Nakamura Y, Yoshimura J. (1991) Synthesis of the functionalized cyclohexanecarbaldehyde derivative. A potential key compound for total synthesis of optically active tetrodotoxin. Chemistry Letters, 1559-1562.

[35] (a) Kim KS, Qian L. (1993) Synthesis of 4,4-difluoro-L-arginine. Tetrahedron Letters, 34, 7195-7196; (b) Bergeron RJ, McManis JS. (1987) Total synthesis of (±)-15-deoxyspergualin. Journal of Organic Chemistry, 52, 1700-1703.

[36] A sample of natural (−)-tetrodotoxin was obtained from Sankyo Co. Ltd. (Tokyo, Japan). [37] Luca LD, Giacomelli G, Porcheddu A. (1975) Chemistry of epoxides. XXXI. Acid-catalyzed reactions of epoxides with dimethyl sulfoxide. Journal

of Organic Chemistry, 40, 2764-2769. [38] (a) Szarek WA, Jewell JS, Szczerek I, Jones JKN. (1969) Addition of pseudohalogens to unsaturated carbohydrates. III. Synthesis of 3-deoxy-3-C-

nitromethyl-D-allose, a branched-chain nitro sugar. Canadian Journal of Chemistry, 47, 4473-4476; (b) Rosenthal A, Sprinzl M. (1969) Branched-chain sugar nucleosides. IV. 9-(3-Deoxy-3-C-hydroxymethyl-β (and α)-D-allofuranosyl and ribofuranosyl) adenine. Canadian Journal of Chemistry, 47, 4477-4481; (c) Kartha KPR. (1986) Iodine, a novel catalyst in carbohydrate reactions. I. O-Isopropylidenation of carbohydrates. Tetrahedron Letters, 27, 3415-3416; (d) Yoshimura J, Sato K, Hashimoto H. (1977) Synthesis of uloses by the oxidation with dimethyl sulfoxide-trifluoroacetic anhydride. Chemistry Letters, 1327-1330; (e) Kinoshita M, Taniguchi M, Morioka M, Takami H, Mizusawa Y. (1988) Synthetic studies of amphotericin B. III. An enantiospecific synthesis of the C(1)-C(19) segment of the amphotericin B aglycon. Bulletin of the Chemical Society of Japan, 61, 2147-2156.

[39] The IUPAC name of compound 44 is 1-L-(1,2,3’,4,5/3,6)-3-hydroxymethyl-3,3’-di-O-benzyl-6-nitro-2,3,4,5-tetrahydroxy-cyclohexancarbaldehyde diphenyldithioacetal.

[40] (a) Otero JM, Barcia JC, Salas CO, Thomas P, Estévez JC, Estévez RJ. (2012) Studies on the Michael addition of naphthoquinones to sugar nitro olefins: first synthesis of polyhydroxylated hexahydro-11H-benzo[a]carbazole-5,6-diones and hexahydro-11bH-benzo[b]carbazole-6,11-diones. Tetrahedron, 68, 1612-1622; (b) Baer H, Rank W. (1965) Reactions of nitro sugars. III. Instability of 6-deoxy-6-nitroaldohexoses at intermediate pH values. Canadian Journal of Chemistry, 43, 3462-3465; (c) Yoshimura J, Funabashi M, Shin C.-G. (1980) Syntheses of a few branched-chain aminocyclitol antibiotics In Aminocyclitol Antibiotics. ACS Symposium Series, Vol. 125, Rinehart Jr. KL, Suami T. (Eds). American Chemical Society, Washington DC, 75-93.

[41] Review of Nef reaction: (a) Ballini R, Petrini M. (2004) Recent synthetic developments in the nitro to carbonyl conversion (Nef reaction). Tetrahedron, 60, 1017-1047; (b) McMurry JE, Melton J, Padgett H. (1974) New method for converting nitro compounds, into carbonyls. Ozonolysis of nitronates. Journal of Organic Chemistry, 39, 259-260.

[42] Review: von Staden LF, Gravestock D, Ager DJ. (2002) New developments in the Peterson olefination reaction. Chemical Society Reviews, 31, 195-200.

[43] Funabashi M, Sato H, Yoshimura J. (1976) Branched-chain sugars. V. Stereoselective synthesis of 3-C- and 5-C-methyl-D-glucoses, and 5-C-methyl-L-idose derivatives. Bulletin of the Chemical Society of Japan, 49, 788-790.

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